Iodine labeled human and rat low-density and high-density lipoprotein degradation by human liver and parenchymal and non-parenchymal cells from rat liver

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Biochimica et Biophysics Acta, 529 (1978) 138-146 @ Elsevier/North-Holland Biomedical Press

BBA 57155

IODINE LABELED HUMAN AND RAT LOW-DENSITY AND HIGH-DENSITY LIPOPROTEIN DEGRADATEON BY HUMAN LIVER AND PARENCHYMAL AND NON-PARENCHYMAL CELLS FROM RAT LIVER THEO J.C. VAN BERKEL, ARIE VAN TOL and JOHAN F. KOSTER Department of Biochemistry (The Netherlands)

I, Erasmus University

Rotterdam,

P.O. Box 1738, Rotter-dam

(Received August 2nd, 1977) (Revised version received December 16th, 1977)

Summary

1. The abilities of homogenates of human liver, rat liver parenchymal cells, rat liver non-parenchymal cells and total rat liver to catabolize human and rat iodinated highdensity lipoprotein (HDL) and lowdensity lipoprotein (LDL) were determined by measuring the amount of trichloroacetic acid-soluble (noniodide) radioactivity liberated upon incubation at the optimum pH of 4.2. 2. A comparison of the capacities of human liver, rat liver and parenchymal and non-parenchymal cells from rat liver indicated that these different preparations are all able to degrade rat iodine-labeled LDL and HDL, with a 5-6-fold higher capacity for HDL as compared to LDL. 3. Iodine-labeled human HDL can be degraded by rat liver, rat parenchymal and rat non-parenchymal cells with a 5-7-fold higher rate than human iodinelabeled LDL. Human liver homogenate was more active in the degradation of both human and rat iodine-labeled LDL and rat HDL as compared to rat liver. 4. A comparison of the capacities of parenchymal and non-parenchymal cells for the degradation of iodine-labeled human and rat LDL and HDL indicates that non-parenchymal cells possess a considerable higher capacity to degrade these lipoproteins per mg of cell protein. 5. The results indicate that a high proportion of the total rat liver capacity for lipoprotein degradation is localized in the non-parenchymal liver cells and this, together with the active endocytic activity, suggests an important role of these liver cells in hepatic lipoprotein catabolism. Introduction Recent evidence in our laboratory [l] ‘has indicated that the enzymes responsible for protein breakdown in the different liver cell types are able to discriminate between low-density and highdensity lipoproteins. This difference in degradation is also found with a total liver supematant (containing supernatant enzymes from both parenchymal and non-parenchymal liver cells) with

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iodinated low-density and highdensity lipoproteins as the substrates [2]. Levy and Eisenberg [3] concluded from a literature survey that the rate of catabolism is an important parameter in the physiological control of the plasma lipoprotein levels. Studies with cultured human fibroblasts [4], aorta smooth muscle cells [5,6] and isolated rat liver parenchymal cells [7] have led to a model for this catabolic process [ 81. This model includes binding of lipoprotein to membrane receptors, followed by active endocytosis and degradation of the proteins and cholesterol esters in the lysosomes by the action of cathepsins and acid lipase, respectively. However, the relative contribution of the various tissues to the in vivo degradation of lowdensity lipoprotein (LDL) and highdensity lipoprotein (HDL) is still controversial. Hay et al. [9] treated male rats with high doses of estrogen and concluded that the liver is the main organ responsible for LDL uptake and degradation. Removal of twothirds of the liver results in a 62% decrease in the fractional catabolic rate [lo] of LDL in the rat. In contrast, Snider-man et al. [ 111 observed an increase in the fractional catabolic rate of LDL after total hepatectomy in the pig. HDL is bound and degraded by isolated rat liver parenchymal cell [7] and the lysosomes have been implicated in the degradation of HDL apoproteins in several species [8,12]. The liver, however, is not a homogeneous tissue and consists of parenchymal cells, which carry out the typical liver functions (e.g. maintenance of the blood glucose level [13]) and non-parenchymal cells (mainly Kupffer cells [14]). Although, on a cellnumber basis, the non-parenchymal cells make up about 35% of the liver cells, their protein contribution to the total liver is only about lo%, due to their smaller size [15]. These non-parenchymal cells can actively take up particles from the blood [16] and are highly enriched with lysosomal enzymes especial cathepsin D and acid lipase [17], two enzymes involved in the proposed model for lipoprotein degradation by cultured fibroblasts [6]. Recently we compared the degradation capacities of total human liver, rat liver and parenchymal and non-parenchymal cells from rat liver to hydrolyze human HDL and LDL by measuring trichloroacetic acid-soluble amino groups equivalents set free under optimal (pH 4.2) conditions [l]. It was found that human as well as rat HDL are degraded by both parenchymal and non-parenchymal cells from rat liver, with a lo-fold higher capacity in the non-parenchymal liver cells. In contrast, human LDL degradation could only be detected in the nonparenchymal liver cells, with a degradation rate relatively low when compared to human HDL. With the applied assay method, however, very high concentrations of lipoproteins were used in order to minimize substantial interference of the homogenate protein with lipoprotein degradation [l,lS]. As the level of serum LDL is very low in the rat, compared to man, it is difficult to compare rat LDL and HDL using this assay procedure. Therefore, we now determined the degradation of LDL and HDL using iodine-labeled lipoproteins. Materials and Methods Isolation and preparation of iodine-labeled lipoproteins Human LDL and HDL * were isolated from human serum in an SW 40 rotor * Human LDL and HDL were isolated from the density ranges 1.019-1.063 and 1.063-1.21 g/ml, respectively. For the rat lipoproteins these ranges were adjusted to 1.019-1.060 and 1.0601.21 g/ml (cf. ref. 20). This modification excludes mutual contamination of rat LDL and HDL (checked by polyacrylamide gel electrophoresis of the delipidated apolipoproteins).

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by the method of Redgrave et al. [ 191. Rat LDL and HDL were isolated from serum of male Wistar rats fed a semi-synthetic high carbohydrate diet for two weeks. The diet was kindly donated by Dr. U.M.T. Houtsmuller (Unilever Research Laboratories, Vlaardingen) and consisted of 72% sucrose, 23% casein and 5% sunflower oil (by calories), supplemented with cellulose, vitamins and minerals. The use of this diet restricted the labeling of lipoprotein phospholipids to less than 4% (see below). The rats were bled under ether anaesthesia after an overnight fast. LDL and HDL were isolated by the same technique as the human lipoproteins. The isolated lipoproteins were iodinated at pH 10 by the ICl method [21], modified for lipoproteins by Langer et al. [22]. Freer was removed by Sephadex G-50 filtration. The iodine : protein ratio was 0.06 and 0.64 atoms/mol for human HDL and LDL and 0.11 and 1.24 atoms/mol for rat HDL and LDL, respectively, assuming an average molecular weight of 20 000 for the apoproteins. 3-4s of the radioactivity in the preparation was free, 3-5s was present in phospholipids and 92-94s was protein bound. Isol4ation 0 f liver cells Isolation of pure and intact rat parenchymal performed as described earlier [ 231.

and non-parenchymal

cells was

Tests for cell purity and integrity The purity and integrity of the rat parenchymal and non-parenchymal cells were determined by phase-contrast microscopy, by trypan blue exclusion and by the relative distribution and specific activities of the L- and M,-type pyruvate kinase isoenzymes. The final parenchymal cell preparations were completely free of non-parenchymal cells and 90% of the isolated cells excluded trypan blue. The non-parenchymal cell preparations excluded trypan blue to more than 95% and were free from parenchymal cells. As shown in Table I, addition of fructose 1,6-bisphosphate to a homogenate of parenchymal cells results, under the applied assay conditions, in a 12-fold stimulation of the pyruvate kinase activity (L-type). No increase in activity is observed with the non-parenchymal cell homogenate indicating its purity. The assay methods for pyruvate kinase have been described earlier [23]. Determination of lipoprotein degradation Freshly isolated cells or livers were frozen

at -80°C

and thawed just before

TABLE I PURITY AND INTEGRITY TEST ON THE ISOLATED CELL PREPARATION DISTRIBUTION OF L- AND M2-TYPE PYRUVATE KINASE Activity in mnol

.

mhrl

. mglprotein

BY MEASURING

(mean t S.E.M. of 4 different preparations).

Source

Activity at 1 mM phosphoenol pyruvate

Activity at 1 mM phosphoenol pynwate + 0.5 mM Fru-1.6-P2

Whole rat liver Parenchymal cells Non-parenchymal cells

24.2 * 2.1 14.0 + 2.6 47.8 * 8.2

157.4 + 9.4 178.7 k 14.2 45.4 + 8.4

THE

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the assays. Homogenization was performed by sonificating twice for 30 s at 21 kHz in a MSE (England) model Cabinet 100 W ultrasonic desintegrator at 0°C. Incubations were carried out for 60 min at 37°C as follows. 20 1.c1of 40 mM dithiothreitol (dissolved in acetate buffer), homogenized cells in 80 ~.tl 0.1 M acetate buffer (pH 4.2) and 100 /.d of iodinated (12’1 or 1311) lipoprotein were mixed at zero time. After 60 min the incubation was stopped by addition of 400 1.1110% trichloroacetic acid. After 15 min at 37°C the mixture was centrifuged for 2 min at 12 000 rev./min (9000 X g). To 400 ~1 of the supematants, 4 ,ul 40% KI and 20 ~1 30% H202 was added. After 5 min at room temperature, 800 ~1 CHC13 were added and the mixture was shaken for another 5 min. After subsequent centrifugation for 20 min at 20 000 rev./min, 200 ~.cl of the aqueous phase (containing iodinated amino acids and small peptides) and 200 /.d of the CHCl, phase (containing I2 formed from I- by oxidation with H,O,) were counted in an LKB-Wallace Ultrogamma counter. In the correspond. ing blanks lipoprotein was added after addition of trichloroacetic acid to the incubated mixtures. Under the applied conditions one CHC& extraction of the aqueous phase resulted in a quantitative removal of CHCl,-extractable radioactivity. Lipoprotein degradation was calculated from the radioactivity measured in the water phase and the specific activities of the protein moieties of the lipoproteins used. It was verified to what extent deiodination of the iodinated amino acids or small peptides formed during the catabolic reaction could lead to an underestimation of the radioactive label in the aqueous phase. For this purpose defatted bovine serum albumin was iodinated with 12’1, by the same method as used for the lipoproteins. 99.3% of the radioactivity in the albumin preparation was protein bound. This iodinated albumin was incubated with the different homogenates under the same experimental conditions as used for the iodinated lipoproteins. After incubation, the CHCIJ phase contained less than 3% of the radioactivity and 97% was recovered from the aqueous phase. This indicates that the radioactivity recovered in the aqueous phase is representative for the degradation of the protein moieties of the lipoproteins and that deiodination under the applied assay conditions is negligible. This is in agreement with data of Stanbury, who showed that the deiodination reaction in liver is located in the microsomes, occurs at neutral pH and needs NADPH as a cofactor [24]. The amount of cell homogenate or liver homogenate protein in the assay varied between 36 and 128 pg. The reaction was found to be linear with this range of protein concentrations and with time up to 4 h under these conditions. The amount of apoprotein added is indicated in the figure and text. The results are expressed as ng apoprotein hydrolyzed min-’ - mg-’ cell homogenate protein. Protein was determined according to Lowry et al. [25].

Hydrolysis of rat lipoproteins Initial studies indicated that the pH optimum for the amount of trichloroacetic acid-soluble radioactivity set free by the incubation of the different preparations with the lipoproteins was between pH 4.0-4.5 (studied pH range, 3.0-9.0). Incubations were performed at pH 4.2 in order to allow a compari-

142 ng protein hydrolyzed

x min

-1

x w

-1

protein

Fig. 1. Hydrolysis of rat iodinated LDL by homogenates of parenchymal cell (PC), non-parenchymal (NPC), total rat liver (RL) and human liver (HL) as a function of the amount of LDL protein @g/ml) Materials and Methods).

cells (see

son with our earlier studies. Fig. 1 shows the degradation rates of iodine-labeled rat LDL as a function of the apoprotein concentration. The curves follow saturation kinetics with half maximal hydrolysis at about 12.5 pg/ml LDL apoprotein. Enzyme(s) saturation is obtained above 100 pg/ml LDL apoprotein with different saturation plateaus for the different rat liver cell types and whole human and whole rat liver. The figure clearly illustrates that the values found with whole rat liver are the result of a contribution of both parenchymal and non-parenchymal cells. To make possible a comparison between the specific capacities (maximal degradation rate/mg of homogenate protein) of the different liver homogenates and cell types to degrade lipoproteins, degradation values must be obtained under enzyme saturation conditions. For this reason Tables II and III show the degradation capacities of the different preparations measured under saturating TABLE

II

DEGRADATION OF IODINE-LABELED BY HUMAN LIVER. RAT LIVER AND RAT LIVER Values are given as ng of lipoprotein S.E.M.. n = 4).

RAT LOW-DENSITY AND HIGH-DENSITY PARENCHYMAL AND NON-PARENCHYMAL

apoproteti

hydrolyzed

mine1

. mg-*

homogenate

LIPOPROTEINS CELLS FROM Protein

(mean

Source

Rat iodlnelabeled LDL

Rat iodine labeled HDL

Whole human liver Whole rat liver Parenchymal cells Non-parenchymal cells Non-parenchymal cell activitylparenchymal

157 ? 11 92 f 10 522 3 389 $ 21 7.5

962 f 48 551 f 42 318 ?: 42 1744 + 138 5.5

Cell activity

+

143 TABLE III DEGRADATION OF IODINE-LABELED HUMAN LOW-DENSITY AND HIGH-DENSITY LIPOPROTEINS BY HUMAN LIVER, RAT LIVER AND PARENCHYMAL AND NON-PARENCHYMAL CELLS FROM RAT LIVER Values are given as ng of lipoprotein apoprotein hydrolyzed S.E.M., n = 4). SOWCI?

Whole human liver Whole rat liver Parenchymal cells Non-parenchymal cells Non-parenchymal cell activlty/parenchymal

cell activity

* miK1 . mg_1homogenate

protein (mean +

Human iodinelabeled LDL

Human iodinelabeled HDL

163 + 10 73k 5 332 3 474 f 57 14.2

441 f 59 363 -’ 52 249 f 42 2376 k 460 9.5

lipoprotein concentrations. Routinely, two apoprotein concentrations were used (between 100 and 250 pg/ml). With these two apoprotein concentrations the same hydrolytic activities were obtained, which ensures enzyme saturation under these conditions for all the cell preparations. As indicated in Table II, a whole rat liver homogenate shows a considerable (6.1-fold) higher capacity for the degradation of rat iodine-labeled HDL when compared to rat iodine-labeled LDL. The same is true for human liver, which is significantly more active in the degradation of rat lipoproteins than rat liver. (For rat LDL P < 0.01 and for rat HDL P< 0.005). The higher capacity of whole rat liver homogenate to degrade HDL as compared to LDL is reflected in both parenchymal and nonparenchymal liver cells (6.1 and 4.5, respectively). Table II further indicates that the non-parenchymal liver cells possess a considerable higher capacity to degrade rat lipoproteins as compared to parenchymal cells with the highest enrichment for rat LDL. From the values obtained with the different rat liver cell types a theoretical value for the whole rat liver can be calculated on the basis of the relative protein contribution to the whole rat liver (90% from parenchymal cells and 10% from non-parenchymal cells). For LDL this value of 0.9 X 52 + 0.1 X 389 = 86 is in good agreement with the measured value, indicating that no apparent changes in protein degradation capacities are introduced by the cell separation procedures. On basis of the relative protein contribution of parenchymal and nonparenchymal liver cells to the whole rat liver homogenate and the capacities found in these different cell types, the importance of the non-parenchymal liver cell capacity for the total capacity of rat liver can be calculated. Such a calculation indicates that 38% of the capacity to degrade rat iodine-labeled HDL and as much as 46% of the capacity to degrade rat iodine-labeled LDL is localized in the non-parenchymal liver cells. Hydrolysis of human lipoproteins Recently [l] we showed, by measuring the amount of trichloroacetic acidsoluble amino groups set free during hydrolysis of human HDL and LDL, that rat liver possesses a higher capacity to degrade human HDL as compared to human LDL. The same difference in degradation rate was found by Stein et al.

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[2] by using iodine-labeled human lipoproteins. Table III shows that a rat whole liver homogenate has a 5-fold higher capacity to degrade human iodinelabeled HDL when compared to human iodine-labeled LDL. However, these data also clearly show that the capacity of human liver to degrade iodinelabeled LDL was significantly greater (P < 0.005) compared to rat liver, resulting in an only 2.7-fold higher capacity of human liver to degrade HDL when compared to LDL. It should be noted that the lysosomal enzymes of the rat parenchymal cells apparently discriminate between human and rat LDL (Tables II and III), leading to a lower degradation capacity for human LDL as compared to rat LDL (P < 0.025) in these cells. It can be calculated from the values given in table III that the total rat liver capacity to degrade human iodine-labeled HDL and LDL is localized in the non-parenchymal liver cells for 51 and 61% respectively, suggesting the importance of these cell types in hepatic lipoprotein catabolism. Discussion Recent progress in the field of lipoprotein catabolism has indicated that the protein moieties of LDL and HDL, after removal from the circulation, are hydrolyzed in the lysosomes probably by the action of cathepsins [ 181. In the present study this hydrolysis was examined in homogenates of human and rat liver and parenchymal and non-parenchymal cells from rat liver under optimal conditions (pH 4.2). The degradation of iodinated LDL and HDL, as measured in the different homogenates, is an enzymatic process with saturation kinetics. For rat iodine-labeled LDL saturation is obtained above 100 pg/ml of apoprotein with half maximal hydrolysis at 12.5 pg/ml. Recently, Stein et al. [ 21 found similar values for human iodine-labeled LDL with total rat liver. Under V conditions we compared the capacities of human and rat liver for the degradation of the various lipoproteins. Rat liver was subdivided into parenchymal cells (90% of liver protein) and non-parenchymal cells, because earlier studies [1,17] indicated that especially the active endo(phago)cytosing nonparenchymal cells are enriched with cathepsin D and acid lipase, resulting in high rates of production of free amino groups from LDL and HDL. The use of iodine-labeled lipoproteins (including rat LDL) enabled us to compare the degradation rates of both rat and human LDL and HDL by rat and human liver. Iodine-labeled lipoproteins provided a sensitive assay with no interference due to the breakdown of tissue proteins. The reported relative rates of degradation of human LDL and HDL by rat whole liver homogenates are in agreement with recent results of Stein et al. [2] who showed a 5.7-fold higher rate for HDL if compared to LDL, while we measured a 5.0-fold difference. For human liver, however, this factor is only 2.7 (due to the significant higher capacity of human liver, as compared to rat liver, to hydrolyze human iodine-labeled LDL (P < 0.005), while the capacities for human iodine-labeled HDL do not differ (P > 0.6)). This indicates that the values obtained for human lipoproteins using rat tissue cannot be simply transferred to human tissue. When we compare the results of this study with the values found earlier [II by using unlabeled HDL and LDL as substrates and measuring the production

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of reactive amino groups under comparable conditions as used in this study, some striking similarities are observed. First, with both methods HDL lipoproteins are degraded much faster than LDL lipoprotein and, second, it appears that non-parenchymal cells show a much higher degradation per mg cell protein than parenchymal cells, although the difference in the present study is smaller for human LDL. This is due to the substantial capacity of parenchymal cell to degrade iodinated LDL (Table III) in contrast to native LDL. This suggests the following possibilities: (1) Native HDL or LDL degradation inhibits the cell protein degradation, leading to underestimated values of reactive amino groups set free during incubation; (2) iodination influences the structural properties of the lipoproteins, leading to a lowering of the discriminating capacity of the parenchymal liver cells. Both possibilities will be the subject of further investigations in our laboratory. Our present and earlier investigations [l] further explore the contribution of the non-parenchymal liver cells to liver lipoprotein catabolism. Studies of Nakai et al. [12] suggest that the liver and liver lysosomes are important organ and subcellular organelle sites for HDL catabolism. However, as recognized by these authors [7], they could not rule out a role of non-parenchymal liver cells in this process. Our present investigations point out that the possible role of the non-parenchymal liver cells cannot be ignored. In fact the non-parenchymal liver cells contribute, 38% for rat HDL, 46% for rat LDL, 51% for human HDL and 61% for human LDL to the total liver capacity for lipoprotein degradation. This high capacity, together with the active endocytosing properties of these cell types might point to an important role of the non-parenchymal liver cells in lipoprotein catabolism. Acknowledgements The authors thank Mr. J.K. Kruijt, Mr. T. van Gent and Miss M.H.M. Verduin for expert technical assistance during the experiments. Professor Dr. W.C. Hiilsmann is thanked for stimulating discussions. Miss A.C. Hanson is thanked for the preparation of the manuscript. The Dutch Heart Foundation is acknowledged for partial financial supprt. References 1 2 3 4 5 6 7 8 9 10 11 12

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